The delivery of therapeutic proteins and peptides in their unmodified form has several limitations, which include poor stability, low solubility, short in vivo circulation, and immunogenicity. Although conjugating proteins and peptides with polyethylene glycol (PEG) -termed PEGylation- is one strategy to overcome these limitations, PEGylation of native proteins or peptides provides limited control of the conjugation site and poor control of stoichiometry because of the promiscuous distribution of common reactive side chains on the surface of most proteins;alternative methods to specify a unique conjugation site on the protein require protein engineering and/or incorporation of "bioorthogonal" unnatural amino acids to create mutant proteins, which is typically a protein specific exercise and alters the composition of the biological drug. Furthermore, in both these approaches, the attachment of presynthesized PEG to the native or mutant protein typically proceeds with low yield because of steric repulsion between these water-soluble macromolecules. Motivated by these limitations, the overall objective of this NIH R01 research proposal is two-fold: the first objective is to develop a new polymer conjugation methodology by the in situ growth of a protein-resistant "stealth" polymer, poly(oligoethylene glycol methacrylate) (POEGMA), exclusively from the N- or C-terminus -the only two conserved reactive sites in a polypeptide sequence- in aqueous buffer by atom transfer radical transfer polymerization (ATRP) with high yield and good control of polymer chain length. We call this methodology InStealth conjugation (in situ growth of stealth polymer), and it yields site- specific stoichiometric conjugates of a PEG-like polymer with tunable molecular weight. The second, related objective is to demonstrate the utility of the InStealth technology to improve the pharmacological profile of a peptide drug -Exendin- that is currently used to treat type II diabetes, but which has fast clearance and poor bioavailability. The impact of these studies will be demonstration of the InStealth conjugation technology as a potentially broadly applicable polymer conjugation technology for the sustained delivery of peptide and protein drugs that overcomes the problems associated with PEGylation. PUBLIC HEALTH RELEVANCE: The proposed research will develop InStealth polymer conjugates, in which a polymer, poly(oligoethylene glycol methacrylate) (POEGMA) will be grown from the C-terminal end of a peptide drug. We will demonstrate the utility of the InStealth technology by improving the efficacy of Exendin, a peptide drug that is used for treatment of type-2 diabetes. We will show that injection of an Exendin-POEGMA conjugate will allow patients to be injected with the drug only once-a-week, as compared to the current twice- daily injections, and that this once-a-week formulation will also result in fewer undesirable side effects.